MMTV-PyMT cells or MC38 cells were seeded in a 96-well tissue culture plate (BD Falcon) at 3000 cells per well and incubated overnight. Cells were washed with DMEM [sodium pyruvate (110 mg/liter), 10% heat-inactivated FBS, 1% penicillin-streptomycin, and Phenol red (−)], and DMEM (80 μl/well) was added. Then, serial dilutions of aldoxorubicin, Dox-SA, or Dox-CBD-SA in PBS were added (20 μl/well). Cells were incubated 3 days at 37°C, and the viability was determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega) according to the manufacturer’s instructions. Cells treated with DMEM (80 μl/well) and PBS (20 μl/well) were defined as 100% viable, whereas the cell-free wells with the same mixture were defined as 0% viable. Half maximal inhibitory concentration (IC50) values were obtained by nonlinear regression analysis in Prism software ([inhibitor] versus normalized response).

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